Hematopoiesis is the process where the body's blood and immune cells are generated from a small number of hematopoietic stem cells (HSCs). These events take place in, and are regulated by, unique parts of the bone marrow termed niches. And while HSCs are responsible for producing nearly one trillion hematopoietic cells a day, mutations in this process can lead to a range of pathologies such as leukemia and bone marrow failure. Further, deficits in homing and engraftment in hematologic disease and during HSC transplant procedures used to treat these diseases significantly reduce patient survival. Previous in vivo and in vitro investigations have identified putative niche components and modes of action, yet culture platforms to predict HSC engraftment, maintain quiescence or direct differentiation do not exist. A major bottleneck is that it remains unclear how the diversit of microenvironmental signals that exist in close spatial and temporal order across the marrow impact HSC response. The goal of this proposal is to use a biomaterial approach to investigate the impact of spatially-organized biophysical signals and marrow-derived niche cells on HSC fate. We will use an engineered bone marrow (EBM) platform to test and revise biophysical hypotheses related to niche regulation of HSC quiescence and activation. To accomplish this goal, we have integrated microfluidic forming tools and orthogonal hydrogel chemistries to generate libraries of optically-translucent 3D biomaterials. Each EBM contains overlapping patterns of marrow-inspired matrix, biomolecule, and cell cues. This approach allows us to isolate small numbers of HSCs from the marrow, establish then manipulate niche signals surrounding these cells in defined increments, and track their response. Uniquely, this system allows selective, stepwise addition of multiple niche-inspired signals in order to reveal their individual versus coordinated impact. Our aims are three-fold. AIM 1: Dissect how overlapping patterns of niche-inspired biophysical signals shape HSC fate. AIM 2: Define the contribution of bone marrow niche cells on quiescence vs. activation. AIM 3: Resolve defects in engraftment and self-renewal for Pcl2-/- HSCs. Leveraging the well-characterized murine hematopoietic system, this approach offers the potential for mechanistic insight regarding native marrow niches, whose rarity and complexity limit direct in vivo examination. This project will generate essential information to aid development of a biomaterial rheostat to control human HSC homeostasis. Such a system would be invaluable for personalized-medicine approaches. As the marrow is the site of leukemogenesis and the target for donor HSC engraftment, EBMs could facilitate more accurate investigations of the etiology, progression, and treatment of hematopoietic diseases. Predictions realized by this approach may also suggest patient-specific improvements in HSC transplant regimens to reduce patient mortality, such as expanding HSCs pre- transplantation vs. pre-conditioning donor niche cells to improve engraftment post-transplantation.